It is well established that enzymes carry out the chemical reactions that are essential to human cell function. We now know that several key enzymes, including the ribosome that carries out protein synthesis, contain essential RNA molecules that participate directly in catalysis. The long-term goal of our research program is to understand how an enzyme active site composed of RNA achieves specificity and chemical rate enhancement comparable to protein enzymes. To pursue this objective we have employed the catalytic RNA, or ribozyme, derived from bacterial ribonuclease P. This ribozyme is highly conserved and homologous to the enzyme found in humans. Thus, it provides an excellent experimental system for exploring fundamental aspects of RNA structure and function. RNase P uses metal ions to promote the attack of a water molecule to break a specific phosphodiester linkage in an RNA precursor to generate a functional tRNA molecule. The proposed research is aimed at providing an understanding of enzyme mechanism, the specific role of metal ions in catalysis, and a functional description of how non-covalent interactions contribute to specificity and rate enhancement. Insight into catalytic mechanism will be gained by examining the effect of substituting heavy isotopes for atoms involved in the chemical reaction. We will begin with an analysis or the attacking water and, in the long term, extend these studies to encompass additional atoms involved in the reaction. We will identify sites of catalytic metal ion binding by examining the effects of substituting specific atoms within the active site of the RNA on reaction rate and metal ion dependence. The role of non-covalent binding interactions will be explored by testing specific models for interactions by making compensatory mutations on the ribozyme and its substrate. Additionally, because enzymes conformation of is generally dynamic, we will extend these studies to determine how changes in conformation are linked to specificity and catalysis. The issues of mechanism addressed are common to all enzymes, thus we expect that the information gained will add to our understanding of biological catalysis. Furthermore, ribozymes, including RNase P, have been adapted to therapeutic purposes, and the results we will obtain should assist in applying catalytic RNAs to this goal.